The Clp1 R140H mutation alters tRNA metabolism and mRNA 3′ processing in mouse models of pontocerebellar hypoplasia

Homozygous mutation of the RNA kinase CLP1 (cleavage factor polyribonucleotide kinase subunit 1) causes pontocerebellar hypoplasia type 10 (PCH10), a pediatric neurodegenerative disease. CLP1 is associated with the transfer RNA (tRNA) splicing endonuclease complex and the cleavage and polyadenylation machinery, but its function remains unclear. We generated two mouse models of PCH10: one homozygous for the disease-associated Clp1 mutation, R140H, and one heterozygous for this mutation and a null allele. Both models exhibit loss of lower motor neurons and neurons of the deep cerebellar nuclei. To explore whether Clp1 mutation impacts tRNA splicing, we profiled the products of intron-containing tRNA genes. While mature tRNAs were expressed at normal levels in mutant mice, numerous other products of intron-containing tRNA genes were dysregulated, with pre-tRNAs, introns, and certain tRNA fragments up-regulated, and other fragments down-regulated. However, the spatiotemporal patterns of dysregulation do not correlate with pathogenicity for most altered tRNA products. To elucidate the effect of Clp1 mutation on precursor messenger RNA (pre-mRNA) cleavage, we analyzed poly(A) site (PAS) usage and gene expression in Clp1R140H/− spinal cord. PAS usage was shifted from proximal to distal sites in the mutant mouse, particularly in short and closely spaced genes. Many such genes were also expressed at lower levels in the Clp1R140H/− mouse, possibly as a result of impaired transcript maturation. These findings are consistent with the hypothesis that select genes are particularly dependent upon CLP1 for proper pre-mRNA cleavage, suggesting that impaired mRNA 3′ processing may contribute to pathogenesis in PCH10.

Ribosome collisions in bacteria promote ribosome rescue by triggering mRNA cleavage by SmrB

Ribosome rescue pathways recycle stalled ribosomes and target problematic mRNAs and aborted proteins for degradation. In bacteria, it remains unclear how rescue pathways distinguish ribosomes stalled in the middle of a transcript from actively translating ribosomes. In a genetic screen in E. coli, we discovered a novel rescue factor that has endonuclease activity. SmrB cleaves mRNAs upstream of stalled ribosomes, allowing the ribosome rescue factor tmRNA (which acts on truncated mRNAs) to rescue upstream ribosomes. SmrB is recruited by ribosome collisions; cryo-EM structures of collided disomes from E. coli and B. subtilis reveal a distinct and conserved arrangement of the individual ribosomes and the composite SmrB binding site. These findings reveal the underlying mechanism by which ribosome collisions trigger ribosome rescue in bacteria.

A Quantitative Arabidopsis IRE1a Ribonuclease-Dependent in vitro mRNA Cleavage Assay for Functional Studies of Substrate Splicing and Decay Activities

The unfolded protein response (UPR) is an adaptive eukaryotic reaction that controls the protein folding capacities of the endoplasmic reticulum (ER). The most ancient and well-conserved component of the UPR is Inositol-Requiring Enzyme 1 (IRE1). Arabidopsis IRE1a (AtIRE1) is a transmembrane sensor of ER stress equipped with dual protein kinase and ribonuclease (RNase) activities, encoded by its C-terminal domain. In response to both physiological stresses and pathological perturbations, AtIRE1a directly cleaves bZIP60 (basic leucine zipper 60) mRNA. Here, we developed a quantitative in vitro cleavage assay that combines recombinant AtIRE1a protein that is expressed in Nicotiana benthamiana and total RNA isolated from Arabidopsis leaves. Wild-type AtIRE1a as well as its variants containing point mutations in the kinase or RNase domains that modify its cleavage activity were employed to demonstrate their contributions to cleavage activity levels. We show that, when exposed to total RNA in vitro, the AtIRE1a protein cleaves bZIP60 mRNA. Depletion of the bZIP60 transcript in the reaction mixture can be precisely quantified by a qRT-PCR-mediated assay. This method facilitates the functional studies of novel plant IRE1 variants by allowing to quickly and precisely assess the effects of protein mutations on the substrate mRNA cleavage activity before advancing to more laborious, stable transgenic approaches in planta. Moreover, this method is readily adaptable to other plant IRE1 paralogs and orthologs, and can also be employed to test additional novel mRNA substrates of plant IRE1, such as transcripts undergoing degradation through the process of regulated IRE1-dependent decay (RIDD). Finally, this method can also be modified and expanded to functional testing of IRE1 interactors and inhibitors, as well as for studies on the molecular evolution of IRE1 and its substrates, providing additional insights into the mechanistic underpinnings of IRE1-mediated ER stress homeostasis in plant tissues.

smartPARE: An R Package for Efficient Identification of True mRNA Cleavage Sites

Degradome sequencing is commonly used to generate high-throughput information on mRNA cleavage sites mediated by small RNAs (sRNA). In our datasets of potato (Solanum tuberosum, St) and Phytophthora infestans (Pi), initial predictions generated high numbers of cleavage site predictions, which highlighted the need of improved analytic tools. Here, we present an R package based on a deep learning convolutional neural network (CNN) in a machine learning environment to optimize discrimination of false from true cleavage sites. When applying smartPARE to our datasets on potato during the infection process by the late blight pathogen, 7.3% of all cleavage windows represented true cleavages distributed on 214 sites in P. infestans and 444 sites in potato. The sRNA landscape of the two organisms is complex with uneven sRNA production and cleavage regions widespread in the two genomes. Multiple targets and several cases of complex regulatory cascades, particularly in potato, was revealed. We conclude that our new analytic approach is useful for anyone working on complex biological systems and with the interest of identifying cleavage sites particularly inferred by sRNA classes beyond miRNAs.

21-nt phasiRNAs direct target mRNA cleavage in rice male germ cells

In grasses, phased small interfering RNAs (phasiRNAs), 21- or 24-nucleotide (nt) in length, are predominantly expressed in anthers and play a role in regulating male fertility. However, their targets and mode of action on the targets remain unknown. Here we profile phasiRNA expression in premeiotic and meiotic spikelets as well as in purified male meiocytes at early prophase I, tetrads and microspores in rice. We show that 21-nt phasiRNAs are most abundant in meiocytes at early prophase I while 24-nt phasiRNAs are more abundant in tetrads and microspores. By performing highly sensitive degradome sequencing, we find that 21-nt phasiRNAs direct target mRNA cleavage in male germ cells, especially in meiocytes at early prophase I. These targets include 435 protein-coding genes and 71 transposons that show an enrichment for carbohydrate biosynthetic and metabolic pathways. Our study provides strong evidence that 21-nt phasiRNAs act in a target-cleavage mode and may facilitate the progression of meiosis by fine-tuning carbohydrate biosynthesis and metabolism in male germ cells.

Nucleocytoplasmic shuttling of Gle1 impacts DDX1 at transcription termination sites

We uncovered a nuclear role for human Gle1 in coordinating transcription termination. When nucleocytoplasmic shuttling of Gle1 is disrupted, nascent mRNA transcripts are elongated. Gle1 colocalizes with DDX1, and loss of Gle1 shuttling impairs recruitment of DDX1 to CstF-64 and transcription termination foci, leading to improper pre-mRNA cleavage.

A functional non-coding RNA is produced from xbp-1 mRNA

The xbp-1 mRNA encodes the XBP-1 transcription factor, a critical part of the unfolded protein response. Here we report that an RNA fragment produced from xbp-1 mRNA cleavage is a biologically active non-coding RNA (ncRNA) in Caenorhabditis elegans neurons, providing the first example of ncRNA derived from mRNA cleavage. We show that the xbp-1 ncRNA is crucial for axon regeneration in vivo, and that it acts independently of the protein-coding function of the xbp-1 transcript. Structural analysis indicates that the function of the xbp-1 ncRNA depends on a single RNA stem; and this stem forms only in the cleaved xbp-1 ncRNA fragment. Disruption of this stem abolishes the non-coding but not coding function of the endogenous xbp-1 transcript. Thus, cleavage of the xbp-1 mRNA bifurcates it into a coding and a non-coding pathway; modulation of the two pathways may allow neurons to fine-tune their response to injury and other stresses.Graphic abstract